BRD4 is being widely investigated as a therapeutic target in inflammatory diseases, as well as a variety of solid and hematological cancers (). It is a chromatin-binding protein with both kinase and acetyl transferase activities that links chromatin structure and transcription and plays an active role in regulating early embryonic development. BRD4 contributes to gene expression in multiple ways, as a scaffold that delivers transcription factors and nucleates superenhancers, as a chromatin remodeler, as a transcription factor that phosphorylates the Pol II CTD. Because deletion of BRD4 is embryonic lethal, the role of BRD4 in normal differentiation has not been extensively investigated in vivo. Consistent with its role as a chromatin remodeler, BRD4-/- thymocytes have significantly reduced levels of acetylated histones necessary for chromatin decompaction and transcription, suggesting the BRD4 plays an active role in thymocyte development. We now have examined the in vivo requirement for BRD4 during thymocyte development. The stages of thymocyte differentiation are characterized by their cell surface expression of the markers CD4 and CD8: DN, ISP, DP and SP. Among these, the ISP subpopulation has been the least well characterized. It has been considered a transitional cell between the DN and DP stages of differentiation. We have found that the ISP represent a discrete subpopulation with a gene expression profile distinct from either the DN precursors or the DP successors. Surprisingly, although BRD4 is expressed throughout all stages of thymocyte differentiation, it selectively affects the differentiation of ISP thymocytes. In contrast to the ISPs, BRD4 is not required during the DN, DP or SP stages of thymocyte development. The maturation of thymocytes is accompanied by large changes in gene expression profiles which are reflected in the patterns of cell surface markers and cell cycle genes. Whereas the DN thymocytes express neither CD4 nor CD8 and are highly proliferative, the ISPs express CD8 and undergo only a single round of proliferation. Accordingly, the transition from DN to ISP is accompanied by large changes in both immune and cell cycle pathways. Interestingly, despite the differences in proliferation, both cell populations express c-myc at approximately equal levels. Thus, myc expression alone does not determine the extent or frequency of proliferation. Although the maturation from ISP to DP is accomplished during a single round of cell cycle, it is accompanied by the differential expression of a large number of genes in immune and metabolic pathways. Unlike either the DN or ISP populations, the DP thymocytes do not express myc, consistent with their quiescent phenotype. Of particular note, the ISP expression profile is distinct from both the DN and the DP. Thus, it is not a transitional population, midway between the DN and DP. Rather, the ISPs are a distinct thymocyte subpopulation. The ISP subpopulation is also distinct in its dependence on BRD4. Although BRD4 is expressed throughout thymocyte differentiation - and at equivalent levels - BRD4 depletion selectively affects the ISP subpopulation. In the absence of BRD4, DN thymocytes differentiate relatively normally, although BRD4-/- DN4 thymocytes are smaller than WT and accumulate in somewhat larger numbers. However, BRD4-/- DN4 thymocytes proliferate and undergo TcRbeta rearrangement normally, reflecting the finding that the expression of only 100 genes is affected. Among the pathways affected are those involved in inflammation and jak-stat signaling. Similarly, BRD4 plays a relatively small role in DP or SP thymocytes which are phenotypically normally and regulates expression of only between 300-400 genes in each cell type which fall into immune system and metabolic processes. In sharp contrast to the modest effects of BRD4 deletion in the DN, DP and SP thymocytes, the ISP are dramatically affected. The expression of over 1100 genes - most in cell cycle and metabolic pathways - is affected. This results in BRD4-/- ISP that are smaller than the WT, unable to undergo cell cycle and deficient in metabolic activity. Importantly, a large fraction of the genes uniquely expressed in ISPs are regulated by BRD4. This leads to the unexpected conclusion that BRD4 is a determining factor in ISP differentiation. BRD4 deletion profoundly affects ISP maturation and progression to the DP stage. Since that progression requires a round of cell cycle, this finding is consistent with the observed loss of the cell cycle regulator c-myc in the absence of BRD4. However, a fraction of the BRD4-deficient ISPs escape and mature to DPs that go on to mature to phenotypically normal SPs. Since no detectable BRD4 remains in the ISP, this raises the question of why not all of the ISPs are affected. One of the major targets of BRD4 in ISP is c-myc. Originally characterized as a specific E-box binding protein, it is now established that c-myc is a general amplifier of transcription that increases expression of genes in proportion to their level of expression. So, in the absence of Myc, many of the same genes would be expressed as in the wild type but at a lower level. This might be enough to stochastically allow some cells, but not others, to mature. However, it also accounts for the escape of a fraction of cells into DP that may reach a threshold of gene expression for cell cycle stochastically. Thus, BRD4 regulates ISP cell cycle through its regulation of myc. Based on our findings, we have proposed a model in which the transition from the highly proliferative DN stage to the quiescent DP stage requires a reprogramming through the ISP stage that is regulated by BRD4. The complete conditional deletion of BRD4 did not allow us to distinguish the roles of its two enzymatic activities in thymocyte differentiation. BRD4 has intrinsic kinase and HAT activities, which have been shown to contribute to the regulation of transcription and chromatin organization, respectively. Replacement of wild type BRD4 with mutants of either kinase or HAT activities results in reduced cell cycle and proliferation of cell lines. We have shown recently that BRD4 phosphorylates MYC, leading to its degradation. Conversely, MYC inhibits BRD4 HAT activity. Therefore, we are generating transgenic mice that express either BRD4 kinase-deficient or HAT-deficient mutant forms to determine the respective roles of each of these activities in differentiation in vivo.